Photosynthetic Responses to Heat Treatments at Different Temperatures and following Recovery in Grapevine (Vitis amurensis L.) Leaves

Background The electron transport chain, Rubisco and stomatal conductance are important in photosynthesis. Little is known about their combined responses to heat treatment at different temperatures and following recovery in grapevines (Vitis spp.) which are often grown in climates with high temperatures. Methodology/Findings The electron transport function of photosystem II, the activation state of Rubisco and the influence of stomatal behavior were investigated in grapevine leaves during heat treatments and following recovery. High temperature treatments included 35, 40 and 45°C, with 25°C as the control and recovery temperature. Heat treatment at 35°C did not significantly (P>0.05) inhibit net photosynthetic rate (P n). However, with treatments at 40 and 45°C, P n was decreased, accompanied by an increase in substomatal CO2 concentration (C i), decreases in stomatal conductance (g s) and the activation state of Rubisco, and inhibition of the donor side and the reaction center of PSII. The acceptor side of PSII was inhibited at 45°C but not at 40°C. When grape leaves recovered following heat treatment, P n, gs and the activation state of Rubisco also increased, and the donor side and the reaction center of PSII recovered. The increase in P n during the recovery period following the second 45°C stress was slower than that following the 40°C stress, and these increases corresponded to the donor side of PSII and the activation state of Rubisco. Conclusions Heat treatment at 35°C did not significantly (P>0.05) influence photosynthesis. The decrease of P n in grape leaves exposed to more severe heat stress (40 or 45°C) was mainly attributed to three factors: the activation state of Rubisco, the donor side and the reaction center of PSII. However, the increase of P n in grape leaves following heat stress was also associated with a stomatal response. The acceptor side of PSII in grape leaves was responsive but less sensitive to heat stress.


Introduction
High temperature negatively affects plant growth and survival and hence crop yield. Photosynthesis is known to be one of the most heat-sensitive processes, and it can be inhibited by high temperature before other symptoms of stress are detected [1,2]. Inhibition of photosynthesis by heat stress has long been attributed to an impairment of electron transport activity, especially the inhibition of photosystem II (PSII) activity [3,4]. Heat stress not only damages the oxygen-evolving complex of PSII [5,6], but also impairs electron transfer within the PSII reaction centres [7,8,9] and downstream of PSII. Some authors [10,11] have suggested that the initial site of the inhibition is associated with a Calvin cycle reaction, especially inactivation of Rubisco [12,13,14,15]. How-ever, for different species, the specific effects of heat stress maybe different.
Worldwide, grape has become one of the most productive and important specialty crops. In many production regions, the maximum midday air temperature can reach more than 40uC, which is especially critical at berry ripening. Some researchers suggested the optimum temperature for photosynthesis is between 25uC and 35uC for some grape cultivars [16,17]. Temperatures above 35uC generally reduce photosynthesis in grape leaves. Climate change may produce more frequent high temperature conditions close to the current northern limit of grape cultivation [18]. Extreme temperatures may therefore endanger berry quality and economic returns [19]. Although there are many reports dealing with the influence of heat stress to photosynthesis in grape leaves [20,21,22,23], a very limited number of papers consider the combined response of the components of PSII, Rubisco and stomatal conductance to heat stress [24,25]. Recently, Kadir [24] and Kadir et al. [25] determined the response of Vitis species to high temperature under controlled environmental conditions through chlorophyll fluorescence measurements such as F v /F m , F v and F o . It is not known if inhibition of grape photosynthesis by heat stress is caused by an impairment of electron transport or Rubisco activity. Moreover, the effect of heat on the donor side, acceptor side, reaction center, and on energy partitioning of PSII in grapevine is not clear. In contrast, studies about effects of low temperatures on photosynthetic performance of grape leaves from electron transport and energy partitioning are relatively abundant [23,26,27]. In addition, recovery from heat stress is an important factor of heat tolerance in plants. Plants, including grapevine, may be most likely to experience heat stress around midday, and relatively normal temperatures otherwise. Thus, plants may be exposed to a heat stress -recovery -heat stress -recovery cycle. However, less information is available on the plant behavior under the heat-stress recovery compared with under heat stress. In the past, attention usually was focused on the plant's direct response to stress. Consequently, more definitive studies on the plant traits for heat tolerance must be conducted to understand the mechanism of the recovery from heat. These results may help develop modern and acceptable technologies to increase and stabilize berry yield and qualities.
Inhibition of PSII leads to a decrease in variable chlorophyll fluorescence. Thus, in vivo chlorophyll fluorescence may be used to detect changes in the photosynthetic apparatus [28,29]. Strasser et al. have developed a method for the analysis of the kinetics of fast fluorescence increases since the non-destructive measurements can be done with a high resolution of 10 ms [30]. All oxygenic photosynthetic materials investigated so far show a polyphasic fluorescence rise consisting of a sequence of phases, denoted as O, J, I, and P (OJIP test). With this test, it is possible to calculate several phenomenological and biophysical expressions of PSII. The kinetics of OJIP are considered to be determined by changes in the redox state of Q A , but at the same time, the OJIP transient reflects the reduction of the photosynthetic electron transport chain [31]. The OJIP test has been a powerful tool for the in vivo investigation of the behavior of PSII function including energy absorption, trapping, and electron transport [30,32,33].
In the present study, gas exchange parameters, chlorophyll fluorescence parameters and the activity state of Rubisco in grape leaves during high temperature (35,40 or 45uC) treatments and following recovery (stress-recovery-stress-recovery) were investigated. Our objective was to determine the importance of electron transport, Rubisco and stomatal factors to maintain photosynthesis and the sensitivity of components of the photosynthetic apparatus in grape leaves under high temperature stress and during recovery.

Results
Net photosynthesis rate (P n ), substomatal CO 2 concentration (C i ) and stomatal conductance (g s ) At normal growth conditions of 25uC, P n , C i and g s of grape leaves did not change during the experiment (over the 3 days of the growth period monitored). Heat stresses at 35uC at two times did not significantly (P.0.05) influence P n , C i and g s of grape leaves compared with the control (at 25uC). A decline of P n and g s after 40 and 45uC treatments was observed, accompanied with a C i increase. Heat stress at 45uC had stronger negative impact on P n and g s than 40uC and recovered more slowly. On the fourth day of recovery (Day 6) after the second heat stress, P n , C i and g s of plants that had received a 40uC treatment recovered to the control levels, but those exposed to 45uC were still exhibiting an effect of heat stress (Fig. 1).

Donor side, reaction centre and acceptor side of PSII and PSI
It has been shown that heat stress can induce a rapid rise in the OJIP polyphasic fluorescence transients. This phase, occurring at around 300 ms and labeled K, is caused by an inhibition of the oxygen evolving complex (OEC). The amplitude of step K can therefore be used as a specific indicator of damage to PSII donor side [26]. Fig. 2 shows the changes in the amplitude in the K step expressed as the ratio W K . Compared with the control (25uC), heat stress at 35uC did not alter W K of grape leaves. After the first heat treatment of 40uC or 45uC for 5 h, W K of grape leaves increased steeply, and W K was higher at 45uC than at 40uC. During the following recovery (on Day 2), W K values of these treatments were similar to the control level. However, they rapidly increased again after the second heat stress. On the first day of recovery (Day 3), they declined to some extent, but W K of the 45uC treatment was bigger than that of the 40uC treatment. On the fourth day of recovery (Day 6), W K of the 40uC and 45uC treatments recovered to the control level.
RC QA shows the density of the of Q A -reducing PSII reaction centers. Fig. 2 demonstrates that heat stress at 35uC did not influence the RC QA during the experiment. The first (on Day 1) and second (on Day 2) stresses of 40uC or 45uC significantly (P,0.05) reduced the RC QA . The RC QA values of the two treatments returned to control values during the first recovery. After the second stress, RC QA values of the two treatments basically reached the level of controls during recovery of the first day. Fig. 3 demonstrates the changes in maximum quantum yield for primary photochemistry (Q Po ), the quantum yield for electron transport (Q Eo ), the probability that a trapped exciton moves an electron into the electron transport chain beyond Q A 2 (y Eo ), the quantum yield for dissipated energy (Q DIo ) in grape leaves during high temperature stress and recovery. Heat stress at 35uC did not significantly (P.0.05) alter Q Po , Q Eo , y Eo and Q DIo in grape leaves. Q Po significantly declined while Q DIo was enhanced at the end of first and second heat stress of 40uC and 45uC. However, at the same time, Q Eo and y Eo showed no change at 40uC, but decreased at 45uC compared with the control. Q Po decreased and Q DIo rose at 40uC less than at 45uC at the first stress. However, Q Po and Q DIo at 40uC was similar to those at 45uC at the second stress. After both stress periods, these parameters recovered to control levels by the first day (Day 2 and Day 3) of recovery.
d Ro expresses the redox state of PSI, i.e., the efficiency with which an electron from PQ through PS I to reduce PS I end electron acceptors. Heat stress at 35uC and 40uC did not change the d Ro in grape leaves, but the d Ro at 45uC rose significantly (P,0.05). However, these parameters recovered to control levels in the first day of recovery (Fig. 4).

PSII efficiency and excitation energy dissipation
PSII efficiency and excitation energy dissipation in grape leaves was examined by modulated fluorescence techniques. Fig. 5 shows that heat stress at 35uC had no effect on the actual PSII efficiency (W PSII ), photochemical quenching coefficient (q p ), as well as nonphotochemical quenching (NPQ). Heat stress at 40 and 45uC led to a sharp decrease of W PSII and q p , and a striking increase of NPQ. After the first 40uC stress, NPQ, W PSII and q p recovered to the control levels the following day (Day 2). With a 1 d recovery after the second 40uC stress, W PSII slowly rose while NPQ declined to some extent, but q p reached the control level. On the fourth day of recovery (Day 6), NPQ, W PSII and q p had recovered to control levels. With the 45uC stress, NPQ, W PSII and q p changed more dramatically, and recovered more slowly after the second stress although they had recovered to the control levels after the first stress.

The activation state of Rubisco
As shown in Fig. 6, heat stress at 35uC had no influence on the activation state of Rubisco in grape leaves compared with 25uC. When the grape leaves were exposed to 40 or 45uC the first time, the Rubisco activation state declined significantly (P,0.05), and 45uC led to the bigger decline. However, after 1 d of recovery, the Rubisco activation state recovered to the control level. When these grape leaves were exposed to 40 or 45uC a second time, the Rubisco activation state declined more than after the first stress, with 45uC resulting in a sharper decrease. On the first day during the second recovery (Day 3), the Rubisco activation state of both treatments had not recovered to the control level although the 40uC treatment recovered more rapidly than the 45uC treatment. On Day 6, the Rubisco activation state of both the 40uC and 45uC treatments reached the control level.

Discussion
The step limiting photosynthesis at high temperatures has been debated recently. One proposed limitation is heat-induced deactivation of Rubisco [12,13,34,35]. The other proposed limitation is impairment of the entire electron transport chain [36,37,38,39]. In fact, different high temperatures may have different effects. This study clearly shows that P n was not limited at 35uC in grape leaves, but it was limited at 40uC and 45uC. This result is similar to views that the optimum temperature for photosynthesis is between 25 and 35uC for some grape leaves [16,17]. When the grape leaves were stressed at 40uC or 45uC, P n and the activation state of Rubisco were markedly reduced while C i increased, indicating that the inhibition of photosynthesis is non-stomatal and associated with Rubisco ( Fig. 1 and 6). The reduction of P n increase proportionally with the increasing of treatment temperature. However, when the grape leaves had recovered from heat stress, the increase of P n was accompanied by increases of g s and the activation state of Rubisco, indicating that P n recovery was also associated with stomatal factors and the activation state of Rubisco ( Fig. 1 and 6). Recent studies with cotton, wheat, tobacco, and maize have confirmed earlier observations that Rubisco is deactivated markedly in response to moderate heat stress [12,13,14,40,41]. However, heat stress at 35uC did not significantly (P.0.05) influence the activation state of Rubisco in grape leaves, which is similar to the effect on P n (Fig. 6). It has been shown that the inhibition of Rubisco activation by moderately elevated temperatures up to 40uC was fully reversible after the heated leaves were incubated at 22.5uC for 15 min [34,42]. In the present study, Rubisco following treatment at 40uC recovered more rapidly than when treated at 45uC.
The decrease of P n under heat stress and increase of P n during recovery was also associated with electron transport capacity. Figs. 2, 3, 4, and 5 show that the PSII and PSI were damaged. In addition, the relationship between P n and electron transport chain was dependent on temperature. Sage and Kubien [43] thought that it has been difficult to pinpoint specific limiting steps that control the temperature response of electron transport chain. However, the OJIP test may be used to demonstrate the limiting steps of electron transport of photosynthesis [44]. At present, the mechanism causing the decline in the electron transport rate above the thermal optimum remains uncertain. Inactivation of the oxygen-evolving complex (OEC) is implicated as a cause of heatinduced reduction in electron transport capacity, particularly at high temperatures (above 38uC in potato and above 40uC in spinach) [3,6]. However, at moderately warm temperatures, this lesion is probably not significant, as leaves can readily alter PSII properties to reduce heat sensitivity of the OEC [3]. The present results showed that 35uC did not result in damage to OEC. Heat stress can influence the PSII reaction center, and the density of RC QA may reflect the density of Q A -reducing PSII reaction centers [45]. In the present study, during the heat stress at 40 or 45uC and the following recoveries, changing trends of W K and RC QA values almost corresponded to that of P n (Fig. 2). This indicated that heat stress and recovery influenced P n partially via the donor side (the oxygen-evolving complex) and reaction center of PSII. Moreover, the higher stress temperature led to a slower recovery of P n .
In these experiments, Q Po declined and Q DIo increased at 40uC or 45uC. However, after 1 d of recovery, they returned to control levels. Interestingly, y Eo and Q Eo significantly (P,0.05) decreased in grape leaves at 45uC but 40uC had almost no influence on Q Eo and y Eo (Fig. 3). Q DIo demonstrates the quantum yield for dissipated energy. In this study, heat stress at 40 or 45uC increased Q DIo . However, Q DIo recovered to control levels after some time. In  addition, d Ro represents the efficiency with which an electron can move from PQ through PSI to the PSI end electron acceptor. The differential response of the d Ro suggests that the redox state of PQpool was affected by the heat stress at 45uC but not 40uC (Fig. 4).
The efficiency of PSII under steady-state irradiance (W PSII ) was closely related to the P n [46]. In this study, under heat stress at 40uC or 45uC W PSII and q p decreased while NPQ increased. This suggests more energy was partitioned to heat dissipation and less energy was used in CO 2 fixation under heat stress at both temperatures. However, the influence at 40uC was less than that at 45uC. After the second recovery, WPSII at 40uC increased more rapidly accompanied by an increase of q p and a decline of NPQ than that at 45uC. A NPQ increase of PSII is widely observed at temperatures where electron transport capacity slows with rising temperature [34,36], which corresponds to the change in Q DIo .

Conclusions
Heat treatment at 35uC did not significantly (P,0.05) influence grapevine photosynthesis. The decrease of P n in grape leaves exposed to heat stress (40 or 45uC) was mainly attributed to the activation state of Rubisco and the donor side and the reaction center of PSII. However, the increase of P n in grape leaves following heat stress was also associated with a stomatal responsec. The acceptor side of PSII in grape leaves was responsive but less sensitive to heat stress.

Plant materials and treatments
One-year old 'Zuoyouhong' grapevines (Vitis amurensis L.) were planted in pots, then grown in a greenhouse at 70-80% relative humidity, 25/18uC day/night cycle, with the maximum photosynthetically active radiation at about 1,000 mmol photons m 2 s 21 .
The progress of the experiment is shown in Table 1. Grapevines with identical growth (10 leaves) were acclimated for two days in a controlled environment room (70-80% relative humidity, 25/ 18uC day/night cycle and 800 mmol m 2 s 21 ) and divided into four groups. On the following day (the first day of the experiment, Day 1), chlorophyll fluorescence and gas exchange parameters were analyzed at 9:30 h for all plants. Then, one group of grapevines was kept at 25uC in this controlled environment room. The other three groups were treated at 35, 40 or 45uC, respectively, in controlled environment rooms (except for temperature, the other conditions were the same as the 25uC room) until 14:30 h, when the relative photosynthesis parameters were then rapidly measured. The stressed grapevines were then allowed to recover at 25uC, with the other conditions the same as before heat treatments. On day 2, the same parameters were measured at 9:30 h, then the grapevines were stressed a second time until 14:30 h, when the relative photosynthesis parameters were then rapidly measured. The treated plants were again allowed to recover at 25uC as above. Chlorophyll florescence and gas exchange parameters were measured at 9:30 h on Day 3 and Day 6 during the following four days of recovery. All of the above measurements were made on the sixth leaf from the top of each plant. The experiment process is in Table 1. Four replications were made with leaves from different grape plants.

Analysis of photosynthetic gas exchange parameters
Photosynthetic gas exchange was analyzed with a Li-Cor 6400 portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA) which can control photosynthesis by means of photon flux density (PPFD), leaf temperature and CO 2 concentration in the cuvette. Net photosynthetic rate (P n ), stomatal conductance (g s ) and substomatal CO 2 concentration (C i ) were determined at a concentration of ambient CO 2 (360 mmol mol 21 ) , a PPFD of   Table 2. Summary of parameters, formulae and their description using data extracted from chlorophyll a fluorescence (OJIP) transient.

Fluorescence parameters Fluorescence parameters Description
Extracted parameters The so-called OJIP-test was employed to analyze each chlorophyll a fluorescence transient by a Plant Efficiency Analyzer (Hansatech Instruments Ltd., King's Lynn, Norfolk, UK) which could provide information on photochemical activity of PSII and status of the plastoquinone pool [48]. The transients were induced by red light of about 3000 mmol photons m 22 s 21 provided by an array of six light emitting diodes (peak 650 nm). The fluorescence signals were recorded within a time span from 10 ms to 1 s with a data acquisition rate of 10 ms for the first 2 ms and every 1 ms thereafter. The fluorescence signal at 50 ms was considered as a true F o . The following data from the original measurements were used: maximal fluorescence intensity (F m ); fluorescence intensity at 300 ms (F k ) [required for calculation of the initial slope (M o ) of the relative variable fluorescence (V) kinetics and W k ]; and the fluorescence intensity at 2 ms (the J-step) denoted as F j , the fluorescence intensity at 30 ms (the I-step) denoted as F i . Terms and formulae are as follows: relative variable fluorescence intensity, V t = (F t 2F o )/(F m 2F o ); a parameter which represent the damage to oxygen evolving complex (OEC), W k = (F k 2F o )/ (F j 2F o ); approximated initial slope of the fluorescence transient, M o = 4(F k 2F o )/(F m 2F o ); probability that a trapped exciton moves an electron into the electron transport chain beyond Q A 2 , y Eo = ET o /TR o = (F m 2F j )/(F m 2F o ); maximum quantum yield of primary photochemistry at t = 0, Q Po = TRo/ABS = F v /F m ; quantum yield for electron transport (at t = 0), Q Eo = ET o / ABS = (F m 2F j )/F m ; quantum yield at t = 0 for energy dissipation,Q DIo = DIo/ABS = F o /F m ; the density of Q A -reducing reaction centers, RC QA = Q Po 6(V j /M o )6(ABS/CS); and the efficiency with which an electron can move from PQ through PSI to the PSI end electron acceptors, dRo = (12V i )/( 12V j ). From OJIP transients, the extracted parameters led to the calculation and derivation of a range of new parameters ( Table 2).
For Rubisco initial activity, a 50 ml sample extract was added to a semimicrocuvette containing 900 ml of an assay solution, immediately followed by adding 50 ml 0.5 mM RuBP, mixing well. The change of absorbance at 340 nm was monitored for 40 s. For Rubisco total activity , 50 ml 0.5 mM RuBP was added 15 min after a sample extract was combined with assay solution to activate all the Rubisco. Rubisco activation state was calculated as the ratio of initial activity to total activity [49,50].

Statistical analyses
Data were processed with SPSS 13.0 for Windows, and each value of the means and standard errors in the figures represents four replications. Differences were considered significant at a probability level of P,0.05 by Duncan's multiple comparison.